Minisequencing on oligonucleotide microarrays: comparison of immobilisation chemistries.
Minisequencing on oligonucleotide microarrays: comparison of immobilisation chemistries.
复制标题
DOI：
10.1093/nar/29.13.e69
复制
发表时间：
2001-07-01
影响因子：
14.9
通讯作者：
Syvanen, A C
中科院分区：
文献类型：
--
作者：
Lindroos, K;Liljedahl, U;Syvanen, A C
在微阵列形式的微测序方法中，使用DNA聚合酶用荧光ddNTP延伸固定在玻璃表面上的多个寡核苷酸引物。该方法是一个有前途的工具，大规模的单核苷酸多态性（SNP）检测。我们比较了八种化学方法共价固定的寡核苷酸引物在玻璃表面上。我们包括市售的活化载玻片和我们自己修改的载玻片。在比较中，评价了不同衍生的载玻片的背景荧光、连接寡核苷酸的效率和微测序反应中引物阵列的性能。我们发现，有显着差异，在不同的涂层之间的背景荧光水平，和附着效率，这是间接测量使用延伸通过末端转移酶，变化很大程度上取决于使用的固定化策略。我们还发现，连接化学影响基因分型的准确性时，微阵列上的微型测序作为基因分型方法。最好的基因分型结果观察使用巯基硅烷包被的载玻片连接二硫键修饰的寡核苷酸。
In the microarray format of the minisequencing method multiple oligonucleotide primers immobilised on a glass surface are extended with fluorescent ddNTPs using a DNA polymerase. The method is a promising tool for large-scale single nucleotide polymorphism (SNP) detection. We have compared eight chemical methods for covalent immobilisation of the oligonucleotide primers on glass surfaces. We included both commercially available, activated slides and slides that were modified by ourselves. In the comparison the differently derivatised glass slides were evaluated with respect to background fluorescence, efficiency of attaching oligonucleotides and performance of the primer arrays in minisequencing reactions. We found that there are significant differences in background fluorescence levels among the different coatings, and that the attachment efficiency, which was measured indirectly using extension by terminal transferase, varied largely depending on which immobilisation strategy was used. We also found that the attachment chemistry affects the genotyping accuracy, when minisequencing on microarrays is used as the genotyping method. The best genotyping results were observed using mercaptosilane-coated slides attaching disulfide-modified oligonucleotides.